We plan to extend our previous studies on mechanisms underlying regulation of Alpha1aARs, by investigating the scaffolding protein spinophilin in Alpha1aAR signaling/ trafficking. Preliminary data demonstrate spinophilin interacts with aiaARs with higher affinity than Alpha1bARs We also plan to extend our studies by studying matnx metalloproteases (MMPs) in mediating Alpha1aAR signaling via EGFR transactivation compared with Alpha1bARs. Here we have a powerful new tool, a.naturally occurnng human 3rd intracellular loop Alpha1a-SNP G247R, which in our preliminary data provides differential activation of specific MMP subtypes Finally, while aims 1 & 2 explore targeted, clinically relevant signaling proteins/ pathways, we recognize the importance of exploring novel binding partners Therefore our last specific aim will use Tandem-Affinity Purification combined with mass spectrometry to identify novel multi-protein complexes (the broader Alpha1aAR signalosome) important in Alpha1aAR signaling/ trafficking.- Unique resources and collaborators available at the Univ of WA make this approach both feasible and quantitative. AIM 1: Targeted examination of the scaffold protein spinophilin in Alpha1aAR signaling and trafficking Hypothesis. Spinophilin binding to the Alpha1gAR 3 rd loop mediates distinct subtype signaling trafficking 1 a Characterize spinophilin/ Alpha1aAR 3rd loop binding (co-immunoprecipitation assays, structure/function analysisryeast 2-hybrid effects) & altered signaling pathways-(specific inhibitors,-siRNAs) 1 b Examine role of spinophilin in Alpha1aAR constitutive & agonist-induced trafficking , 1 c. Compare Alpha1aAR sphinophilin-modulated signaling effects with OibAR and Alpha1aAR 3rd loop SNP G247R AIIM 2: Investigate role of IMMPs in EGFR transactivation in Alpha1aAR signaling Hypothesis: Differential activation of specific MMPs by Alpha1AR subtypes mediates distinct signaling /trafficking 2 a Characterize role of MMPs in EGFR transactivation for differential Alpha1aAR signaling (WT vs G247SNP) 2 b Examine gene expression arrays for OigAR WT ve'rsus G247 SNP & aib to identify specific MMPs involved (confirm using specific transactivation assays and inhibitors) AIM 3' Utilize genomic/proteomic approaches to identify broader Alpha1aAR signalosome Hypothesis. Proteomics can be used to identify novel proteins in broader multi-protein complexes (signalosome) important in unique Alpha1gAR signaling and cellular trafficking 3.a Create tandem-affinity purification (TAP)-tag constructs for OigAR, OibAR Oia-SNP, establish cell lines 3.b Use proteomics-tandem reverse-phase liquid chromatography mass spectrometry (LC-MS/MS, LTQFT) technology to identify multi-protein complexes important in Alpha1a versus Alpha1bAR signaling/trafficking 3 c Perturb system (agonist/antagonist drugs, G247R Alpha1-SNP) to identify differential signalosomes' 3 d Compare Alpha1aAR signalosomes in 4 cell lines commonly used in Alpha1aAR studies (rat/human with different surface versus intracellular receptor expression to identify key proteins modulating Alpha1aAR trafficking